Dimerization mechanisms of two procaspase subfamilies

描述(由申请人提供)： 调节原天冬氨酸氨基转移酶激活的机制在细胞凋亡和炎症的调节中起着核心作用。研究表明，原天冬氨酸氨基转移酶二聚体的形成是成熟过程中的一个关键事件。例如，proaspase-1被认为是一种单体，直到caspase招募结构域(CARD)中的相互作用驱动蛋白酶域的二聚化。支架足以进行自溶处理。相反，我们已经证明proaspase-3是一种稳定的二聚体，即使它不包含卡片。这提示了激活原天冬氨酸酶-1和-3的不同折叠和调节机制。我们假设二聚体界面的不同是蛋白质是单体还是二聚体的关键。此外，我们还证明了二聚化作用与酶活性之间存在联系。基于我们的蛋白质工程研究，我们假设蛋白质稳定性和酶活性的提高是通过一个从二聚体界面延伸到两个活性位点的氨基酸网络来实现的。我们认为原天冬氨酸酶作为分子机器，在其中二聚体界面上的侧链运动影响活性部位的运动，从而允许底物结合口袋的形成。我们将通过解决以下具体目标中的三个关键问题来处理这一问题。1.proaspase-1和-3是否通过相似的机制折叠和组装？已建立的生物物理方法将被用来确定proaspase-1的寡聚体性质，以测试当前的范式。2.二聚作用与活性中心的形成是如何联系的？利用蛋白质工程技术，我们将研究二聚体界面附近四个位置上氨基酸的明显连接，以及影响活性位点环正确插入的活性位点。3.前结构域在折叠和组装中是如何发挥作用的？有证据表明，该前多肽具有分子内伴侣的功能。将利用分子生物学和生物物理研究来确定确切的作用机制。这项工作有可能影响一些自身免疫性疾病、利尔特病和癌症的治疗策略，因为细胞凋亡是这些疾病的共同因素。学习有选择地操纵细胞凋亡的水平很可能会导致这些疾病的治疗策略。

DESCRIPTION (provided by applicant): The mechanisms that regulate the activation of procaspases play central roles in the regulation of apoptosis and inflammation. It has been shown that formation of a procaspase dimer is a critical event in maturation. For example, procaspase-1 is thought to be a monomer until interactions in the caspase recruitment domain (CARD) drive dimerization of the protease domains. The scaffold is sufficient to allow autolytic processing. In contrast, we have shown that procaspase-3 is a stable dimer, even though it does not contain a CARD. This suggests different folding and regulatory mechanisms for the activation of procaspases-1 and -3. We hypothesize that differences in the dimer interfaces are the key to whether the protein is a monomer or dimer. In addition, we show that dimerization and enzymatic activity are linked. Based on our protein engineering studies, we hypothesize that the gains in protein stability and enzyme activity are linked via a network of amino acids that extends from the dimer interface to the two active sites. We suggest that procaspases act as molecular machines in which side chain movements in the dimer interface affect the movements in the active site, allowing the substrate-binding pocket to form. We will approach this problem by addressing three key questions in the following specific aims. 1. Do procaspases-1 and -3 fold and assemble via similar mechanisms? Established biophysical methods will be employed to determine the oligomeric properties of procaspase-1 in order to test the current paradigm. 2. How is dimerization linked to active site formation? Using protein engineering techniques, we will examine the apparent linkage of amino acids at four positions near the dimer interface and active sites that affect proper insertion of the active site loops. 3. How does the pro-domain function in folding and assembly? Evidence is presented that the pro-peptide functions as an intramolecular chaperone. Molecular biological and biophysical studies will be employed to determine the precise mechanism of action. This work has the potential to affect therapeutic strategies for a number of autoimmune diseases, leart disease, and cancers because apoptosis is a common factor to these diseases. Learning to electively manipulate the level of apoptosis may well lead to therapeutic strategies for these diseases.